This grant will continue to support our ongoing discovery antd functional characterization of molecular mechanisms of lymphocyte homing to the Gl tract. We propose the following specific Aims: Aim 1. GPR15 in intestinal immunobiology: role in intestinal immune responses and mechanisms and effects of species specific differences in expression. We showed expression of GPR15 by effector T cells (Teff) in colitis, and demonstrated that this novel colon homing receptor can play a pathogenic role. We also discovered major differences in GPR15 expression in humans vs mice that have the potential to underlie differences in inflammation, vaccination responses, and intestinal immunity. We propose that aberrant GPR15 expression in mice is due to absence of GATA3 consensus binding sites that regulate GPR15 in other species. We will use CyTOF and flow cytometry to compare the expression and regulation of GPR15 on gut homing lymphocyte subsets in mouse vs man. We will use standard reporter assays, site directed mutagenesis and Chip-seq data to test the hypothesis that the species differences in Th vs Treg subset expression are controlled by loss of GATA3 binding to the mouse Gpr15 enhancer. Most importantly, we propose to analyze the effects of humanized Gpr15 expression patterns on small intestinal Teff vs Treg subsets, and on intestinal immune responses in vivo. Mice in which GPR15 is driven under native human sequence control (BAG transgenics; 5 founder lines have been generated already), or in which GATA3 TFBS are introduced or repaired in the mouse enhancer sequence by targeted mutagenesis or knockin strategies, will be studies under homeostatic and inflamed/immunized conditions. The effects of altered, 'human-like' GPR15 expression patterns on intestinal lymphocyte subset frequencies and immune responses will be assessed. Significance: The studies will directly test the hypothesized role of GATA3 TFBS mutations in human vs mouse differences in GPR15 expression and in intestinal immunobiology. If, as hypothesized, mice with a humanized Ggr15 enhancer display colon immunobiology that much more closely reflects that of humans, the studies will have fundamental implications for our understanding of colitis and for drug discovery for IBD. Aim 2: To define the role and significance of CD22 as a novel intestinal lymphocyte homing receptor Our experiments show that CD22 functions as a B cell homing receptor for HEV in Peyer's patches. Short term homing assays will be used to confirm this, and to assess critically a hypothesized role of CD22 in homing of follicular T helper cell subsets to GALT as well. We will also assess the role of CD22 in lymphocyte recruitment to the lamina propria and colon patches. Finally, we observe significant residual Sfga/6-dependent homing of CD22-deficient lymphocytes to PP: we hypothesize that this may be mediated by the closely related B cell lectin, SiglecG, which binds similar carbohydrates. We propose to use readily available SiglecG and if indicated, CD22/SiglecG double KO mice to test this hypothesis. Significance: This Aim will elucidate specific B cell homing mechanisms for GALT, mechanisms that contribute to the specialization of mucosal immune responses. Moreover, CD22 is an emerging target for autoimmune diseases: Understanding its role in lymphocyte recruitment to the Gl tract will provide important insight into intestinal immune responses, and could prove critical to avoid infectious side effects in settings of CD22 targeted immunotherapy. Aim 3: GPR43 in intestinal immunity and immune homeostasis: role in Th17 vs Treg homeostasis, and identification and function of novel protein ligands. Based on our preliminary studies, we propose to use GPR43 KO /WT mixed bone marrow chimeras to define the role of the receptor in microbiome- and short chain fatty acid (SCFA)-dependent control of Th17 and other effector and regulatory T cells in the gut. In addition, we will elucidate GPR43 interactions with angiopoietin-2 (Ang2). In the DSS colitis model Ang2, like GPR43, reduces the severity of symptoms (colon shortening, diarrhea), although the effects are complex. We shall use standard biochemical assays to define the specificity/avidity of Ang2-GPR43 binding, and in vitro adhesion assay to define the role of Ang2 in intestinal immune cell adhesion triggering and chemotaxis. Since GPR43 regulates apoptosis, we will also elucidate the effects of Ang2-GPR43 interaction on immune cell survival and responses to pro-inflammatory signals. Significance: GPR43 moderates inflammation in the Gl tract, dampening colitis. Its known ligands are short chain fatty acids (SOFA) from intestinal microflora. Identification of Ang2 as a protein ligand and for GPR43 suggests the potential for additional pathways to GPR43-dependent control of intestinal immune responses. Since Ang2 regulates angiogenesis and vascular stability, its induction during gut wall repair may act through GPR43 to suppress inflammation in the absence of dangerous pathogen invasion. Additional Plans: We will test the hypothesized roles of additional candidate trafficking mechanisms identified in our microarrays. Anti-CD63 and Bsti MAbs will be injected iv to assess effects on lymphocyte homing to GALT and gut wall. To test the hypothesis that GPR183 mediates lymphocyte diapedesis in response to a trans-HEV gradient of its oxysterol ligands, we will compare the in vivo transendothelial migration and homing ability of normal B and T cells to those of lymphocytes in which GPR183 is deficient or desensitized. Significance: These studies have the potential to define additional mechanisms and therapeutic targets for regulation of intestinal lymphocyte trafficking.